This invention relates to the novel identification of myelin-associated glycoprotein (xe2x80x9cMAGxe2x80x9d) as a potent inhibitor of neural regeneration. More particularly, this invention relates to compositions and methods useful for reversing inhibition of neural regeneration in the central and peripheral nervous system. Assays to monitor the effects of MAG on neural regeneration and to identify agents which will block or promote the inhibitory effects of MAG on neural outgrowth are provided. Screening methods for identifying such agents are also provided. This invention also relates to compositions and methods using agents that can reverse the inhibitory effects of MAG on neural regeneration. Methods for regulating and for promoting neural growth or regeneration in the nervous system, methods for treating injuries or damage to nervous tissue or neurons, and methods for treating neural degeneration associated with disorders or diseases, comprising the step of administering at least one of the compositions according to this invention are provided.
The mammalian nervous system does not regenerate after injury despite the fact that there are many molecules present which encourage/promote axonal (nerve) growth. It is believed that the lack of regeneration caused by the presence of molecules in the central nervous system (CNS) and the peripheral nervous system (PNS) which actively prevent/inhibit regeneration. Hence, the well documented inability of the adult mammalian CNS to regenerate after injury is believed to result from a predominance of inhibitory molecules.
It has been demonstrated that when neurons are grown on tissue sections of the CNS they fail to extend processes onto areas of white matter, myelin. It is believed that myelin-specific inhibitory molecules can largely account for the lack of CNS regeneration and their identification will help in the design of therapies to encourage regrowth after injury. The precise molecules responsible for this inhibition have, so far, remained elusive. If these inhibitory molecules can be identified and blocked, then neural regeneration can be encouraged.
Schwab and co-workers have identified two components in CNS myelin, in the molecular weight ranges of approximately 35 kD and 250 kD, which arrest axonal growth. The most compelling observation in support of the inhibitory action of these two protein fractions is that antibodies raised to proteins eluted from these regions of polyacrylamide gels after separation of CNS myelin proteins, specifically reverses the inhibitory effect of myelin in vitro and allows limited spinal cord regeneration when applied in vivo to transected nerves (Caroni, P. and Schwab, M. E., Neuron, 1, pp. 85-96 (1988a); J. Cell Biol., 106, pp. 1281-88 (1988b); Schnell, L. and Schwab, M. E., Nature, 343, pp. 269-72 (1990)). The nature of these two proteins and how they act have not yet been described, but, it is generally accepted that they are significant contributors to the inhibitory effect of this tissue. However, as acknowledged by the authors, other factors are likely to contribute to the inhibition by CNS myelin as even in the presence of antibodies directed against these two proteins, the majority of axons in vivo fail to regenerate (Schnell, L. and Schwab, M. E., Nature, 343,pp. 269-72 (1990); Schnell et al., Nature, 367,pp. 170-73 (1993)).
In addition to inhibitory molecules in myelin, another family of proteins has recently been identified whose members inhibit axonal regeneration. These molecules are called collapsins (Luo et al., Cell, 75,pp. 217-27 (1993)). However, collapsins are found ubiquitously throughout the nervous system and as they are found in regions of the nervous system in which axons will grow, i.e. gray matter, they are unlikely to contribute significantly to the lack of neural regeneration after injury. Instead, the collapsins most likely play a role in guiding growing axons during development.
Previously it was shown that MAG, like many members of the Ig-superfamily of molecules, could promote neurite outgrowth, in this case, from dorsal root ganglion (DRG) neurons from 2 day old rats (13). We observed a similar effect on DRG neurons from rats up to postnatal day 3, but after this age MAG had the opposite effect, i.e., it inhibited neurite outgrowth (Mukhopadhyay et al., Neuron, 13, pp. 757-67 (1994)). Furthermore, we also found that MAG dramatically inhibited neurite outgrowth from cerebellar neurons from rats of all ages up to adult. Polyclonal antibodies directed against MAG could specifically block both stimulatory and inhibitory effects of MAG on neurite outgrowth. MAG, therefore, depending on the age and the type of neuron, can either promote or inhibit neurite outgrowth. Subsequent to our report on the inhibitory effects of MAG, another group demonstrated, using a different complementary approach, that MAG is an inhibitor of axonal growth (McKerracher et al., Neuron, 13, pp. 805-811 (1994); WO 95/22344 (Aug. 24, 1995); incorporated herein by reference).
It would be useful to block the inhibitors of axonal regeneration for treating patients with nervous system injuries where neural regeneration is a problem. No molecule had been identified in myelin which is a potent inhibitor of axonal regeneration. Although Schwab and co-workers identified components in myelin that are inhibitory, the precise nature of these components has not been identified, i.e., they have not been cloned nor have the proteins been purified. In addition, there was no information available on the component on the neuron that the putative inhibitory molecules interact with to prevent regrowth. As no inhibitory nor interacting molecules had been precisely identified, it was difficult, if not impossible, to logically design strategies whereby these molecules can been blocked and prevented from inhibiting neural regeneration.
The present invention solves the problems referred to above by identifying MAG as a potent inhibitor of axonal regeneration in the central nervous system (CNS) and the peripheral nervous system (PNS). The present invention provides compositions and methods for blocking or manipulating the levels of MAG activity in the nervous system.
In one embodiment, the compositions comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one inhibitor of MAG. Inhibitors of MAG include but are not limited to anti-MAG antibodies, altered and/or mutated forms of MAG characterized by an altered biological activity, free sialic acid-bearing sugars, modified derivatives of sialic acid attached to a sugar, a sialic acid-bearing sugar attached to a protein or lipid carrier molecule, a modified sialic acid-bearing sugar attached to a protein or lipid carrier molecule and a sialic acid glycopeptide.
In one preferred embodiment, the MAG inhibitor comprises a small sialic acid-bearing oligosaccharide (sugar), which is optionally a competitive inhibitor of sialidase. More preferably, the sialic acid analog is sialo 2,3-xcex1 lactose (2,3-SL) or 2,3-dideoxy sialic acid (DD-NANA).
In another preferred embodiment, the MAG inhibitor comprises an altered and/or mutant form of MAG which can inhibit the binding of endogenous MAG to neurons in the CNS or PNS. Altered forms of MAG preferably comprise all or a portion of the extracellular domain of MAG fused to another molecule which renders the chimeric protein soluble. One such preferred soluble MAG chimeric protein comprises the five Ig-like domains of MAG fused to the Fc domain of a human immunoglobulin molecule, such as IgG (xe2x80x9cMAG-Fcxe2x80x9d).
Preferred altered/mutated forms of MAG are soluble molecules which harbor one or more mutations in the MAG molecule that reduce or eliminate its ability to inhibit or promote neurite outgrowth compared to endogenous MAG or MAG-Fc, but do not significantly diminish the binding of the altered or mutant form of MAG to neuronal surfaces. Most preferred altered/mutant forms of MAG are soluble molecules comprising a truncated form of MAG-Fc consisting of the first three of the five extracellular Ig-like domains of MAG fused to an immunoglobulin Fc domain (xe2x80x9cMAG(d1-3)-Fcxe2x80x9d).
In another embodiment, the compositions comprise a therapeutically effective amount of an enzyme that can alter or remove sialic acid residues having a Neu5Acxcex12xe2x86x923Galxcex21xe2x86x923GalNAc (3-O) structure, which mediate MAG binding to neuronal surfaces in the PNS or CNS. Preferred compositions of this embodiment comprise sialidase (a neuraminidase) and sialyl transferases that alter the structure and/or lower the effective concentration of Neu5Acxcex12xe2x86x923Galxcex21xe2x86x923GalNAc (xe2x80x9c3-Oxe2x80x9d) sialyated glycans.
The present invention also provides methods for regulating and for promoting neural growth or regeneration in the nervous system, methods for treating injuries or damage to nervous tissue or neurons, and methods for treating neural degeneration associated with disorders or diseases, comprising the step of administering at least one of the pharmaceutical compositions according to this invention.
The present invention provides an assay for determining whether neurite outgrowth from a particular type of neuron at a particular age is stimulated or inhibited in the presence of MAG (or a MAG derivative). In one embodiment, the method comprises the steps of:
a) culturing a first sample of a selected neuronal cell type on a growth-permissive substrate in the absence of MAG;
b) culturing a second sample of the selected neuronal cell type on a growth-permissive substrate comprising bound MAG; and
c) comparing the relative amount of neurite growth in the cultured cells of a) and b); wherein when the relative growth of neurites in the cultured cells of a) is greater than in b), the neuronal cell is inhibited by the presence of MAG, and when the relative growth of neurites in the cultured cells of a) is less than in b), the neuronal cell is stimulated by the presence of MAG.
In a preferred embodiment, the growth-permissive substrate in the absence of MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG, and the growth-permissive substrate comprising bound MAG comprises a monolayer equivalent mammalian cells engineered to express cell surface MAG. Preferably, the mammalian cells are CHO cells engineered to express cell surface MAG, such as CHO-MAG2 cells.
The present invention also provides methods for identifying a MAG-dependent neurite growth altering agent, i.e., an agent which alters neurite outgrowth from a selected neuronal cell type, or population of mixed cell types, in the presence of MAG compared to the absence of MAG.
In one embodiment, the method comprises the steps of:
a) culturing a first sample of a selected neuronal cell type on a growth-permissive substrate in the absence of MAG;
b) culturing a second sample of the selected neuronal cell type on a growth-permissive substrate comprising bound MAG in an amount sufficient to alter neurite outgrowth from the cells compared to the first sample of cells cultured in the absence of MAG;
c) incubating the cell cultures of a) and b) with a known relative concentration of a test agent for a time sufficient to allow neurite growth; and
d) comparing the relative amount of neurite growth in the cultured cells of a) and b); wherein an agent that changes the relative growth of neurites in the cultured cells of a) and b) is identified as a MAG-dependent neurite growth altering agent.
In a preferred embodiment, the growth-permissive substrate in the absence of MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG, and the growth-permissive substrate comprising bound MAG comprises a monolayer equivalent mammalian cells engineered to express cell surface MAG. Preferably, the mammalian cells are CHO cells engineered to express cell surface MAG, such as CHO-MAG2 cells.
In another embodiment of this invention, the method for identifying a MAG-dependent neurite growth altering agent comprises the steps of:
a) culturing separate samples of a selected neuronal cell type on a growth-permissive substrate lacking MAG;
b) culturing a first sample of a) with a known concentration of a traceable, soluble form of MAG;
c) culturing a second sample of a) with a known concentration of a traceable, soluble form of a control protein lacking MAG activity;
d) incubating the cultures of b) and c) with a known relative concentration of a test agent for a time sufficient to allow neurite growth; and e) comparing the relative amount of neurite growth in the cultured cells of c) and d); wherein an agent that changes the relative growth of neurites in the cultured cells of c) and d) is identified as a MAG-dependent neurite growth altering agent.
In one preferred embodiment, the growth-permissive substrate lacking MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG, such as COS or NIH 3T3 cells. In another preferred embodiment, the growth-permissive substrate lacking MAG comprises an immobilized monolayer of a purified, growth-promoting factor. One most preferred neuronal growth-promoting factor which may be immobilized onto a monolayer is the L1 glycoprotein.
In preferred embodiments, the soluble form of MAG is a MAG-Fc fusion protein, and the soluble control protein lacking MAG activity is a MUC 18-Fc fusion protein. Preferred traceable fusion proteins are radioactively or fluorescently labeled.